Gap Junction Protein Cx43 Research Articles

Increased Cardiac Arrhythmogenesis Associated With Gap Junction Remodeling With Upregulation of RNA-Binding Protein FXR1.

Gap junction remodeling is well established as a consistent feature of human heart disease involving spontaneous ventricular arrhythmia. The mechanisms responsible for gap junction remodeling that include alterations in the distribution of, and protein expression within, gap junctions are still debated. Studies reveal that multiple transcriptional and posttranscriptional regulatory pathways are triggered in response to cardiac disease, such as those involving RNA-binding proteins. The expression levels of FXR1 (fragile X mental retardation autosomal homolog 1), an RNA-binding protein, are critical to maintain proper cardiac muscle function; however, the connection between FXR1 and disease is not clear. To identify the mechanisms regulating gap junction remodeling in cardiac disease, we sought to identify the functional properties of FXR1 expression, direct targets of FXR1 in human left ventricle dilated cardiomyopathy (DCM) biopsy samples and mouse models of DCM through BioID proximity assay and RNA immunoprecipitation, how FXR1 regulates its targets through RNA stability and luciferase assays, and functional consequences of altering the levels of this important RNA-binding protein through the analysis of cardiac-specific FXR1 knockout mice and mice injected with 3xMyc-FXR1 adeno-associated virus. FXR1 expression is significantly increased in tissue samples from human and mouse models of DCM via Western blot analysis. FXR1 associates with intercalated discs, and integral gap junction proteins Cx43 (connexin 43), Cx45 (connexin 45), and ZO-1 (zonula occludens-1) were identified as novel mRNA targets of FXR1 by using a BioID proximity assay and RNA immunoprecipitation. Our findings show that FXR1 is a multifunctional protein involved in translational regulation and stabilization of its mRNA targets in heart muscle. In addition, introduction of 3xMyc-FXR1 via adeno-associated virus into mice leads to the redistribution of gap junctions and promotes ventricular tachycardia, showing the functional significance of FXR1 upregulation observed in DCM. In DCM, increased FXR1 expression appears to play an important role in disease progression by regulating gap junction remodeling. Together this study provides a novel function of FXR1, namely, that it directly regulates major gap junction components, contributing to proper cell-cell communication in the heart.

Abstract 198: Multipotent Placenta-derived Cdx2 Cells Possess in vitro and in vivo Cardiomyogenic Potential

Stem cell-based therapies for cardiac regeneration are of crucial importance and an ideal cell-type is yet to be established. We previously reported that fetal cells from placenta “home” to injured maternal heart and approximately 40% (40/100) of the migrating cells expressed homeodomain protein Cdx2. This interesting observation led us to hypothesize that placental Cdx2 could be a novel cell target for cardiac differentiation. To understand this phenomenon, we employed a cre-lox strategy that labeled Cdx2 cells in placenta with e-GFP and induced myocardial infarction (MI) in pregnant mice at mid-gestation. The maternal heart was analyzed 4 weeks post-MI for the presence of Cdx2-eGFP-derived cardiomyocytes. Additionally, Cdx2 cells were isolated from late-gestation placenta and assayed for cardiac differentiation in vitro followed by live cell imaging. Phenotypic and whole-cell proteomic analysis, clonal and vascular lineage differentiation and immune profiling were carried out subsequently. We observed that Cdx2 cells migrated to injured maternal hearts and differentiated into cardiomyocytes highlighting the functional significance of fetal-maternal stem cell transfer. Additionally, isolated Cdx2 cells from the late placenta differentiated into spontaneously beating cardiomyocytes and expressed structural proteins cardiac troponin T(cTnT), α-sarcomeric actinin and gap junction protein Cx43. These cells underwent clonal expansion and differentiated into endothelial and smooth muscle lineages in culture indicative of their multipotent nature. Low expression of MHC molecules and other components of the immune-response, infer that these cells possess the ability to evade host immune surveillance. Proteomic analysis demonstrated that 145 proteins were uniquely identified in the Cdx2 cells compared to embryonic stem cells. These protein networks reflected an increased activation of functions involving migration, fertility, homing, and chemotaxis. Our study is the first to demonstrate that Cdx2 may play a role in cardiac differentiation and delineate multipotent cells in placenta with an inherent “homing” ability. These findings point to a potential role for Cdx2 cells in cardiac regenerative therapies using allogeneic cells.

Cx43 suppresses evx1 expression to regulate joint initiation in the regenerating fin.

How joints are correctly positioned in the vertebrate skeleton remains poorly understood. From our studies on the regenerating fin, we have evidence that the gap junction protein Cx43 suppresses joint formation by suppressing the expression of the evx1 transcription factor. Joint morphogenesis proceeds through at least two discrete stages. First, cells that will produce the joint condense in a single row on the bone matrix ("initiation"). Second, these cells separate coincident with articulation of the bone matrix. We propose that Cx43 activity is transiently reduced prior to joint initiation. We first define the timing of joint initiation with respect to regeneration. We next correlate reduced cx43 expression and increased evx1 expression with initiation. Through manipulation of cx43 expression, we demonstrate that Cx43 negatively influences evx1 expression and joint formation. We further demonstrate that Cx43 activity in the dermal fibroblasts is required to rescue joint formation in the cx43 mutant, short finb123 . We conclude that Cx43 activity in the dermal fibroblasts influences the expression of evx1, and therefore the differentiation of the precursor cells that give rise to the joint-forming osteoblasts. Developmental Dynamics 246:691-699, 2017. © 2017 Wiley Periodicals, Inc.

Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling.

Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin-deficient (CD11b-/-) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo-/-) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo-/- mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell-derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo-/- mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter-defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.

Cx43 Mediates Resistance against MPP⁺-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells via Modulating the Mitochondrial Apoptosis Pathway.

Neuronal apoptosis in the substantia nigra par compacta (SNpc) appears to play an essential role in the pathogenesis of Parkinson’s disease. However, the mechanisms responsible for the death of dopaminergic neurons are not fully understood yet. To explore the apoptotic mechanisms, we used a well-known parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP+), to induce neuronal apoptosis in the human dopaminergic SH-SY5Y cell line. The most common method of interaction between cells is gap junctional intercellular communication (GJIC) mediated by gap junctions (GJs) formed by transmembrane proteins called connexins (Cx). Modulation of GJIC affects cell viability or growth, implying that GJIC may have an important role in maintaining homeostasis in various organs. Here, we hypothesized that increasing the level of the gap junction protein Cx43 in SH-SY5Y neuroblastoma cells could provide neuroprotection. First, our experiments demonstrated that knocking down Cx43 protein by using Cx43-specific shRNA in SH-SY5Y neuroblastoma cells potentiated MPP+-induced neuronal apoptosis evident from decreased cell viability. In another experiment, we demonstrated that over-expression of Cx43 in the SH-SY5Y cell system decreased MPP+-induced apoptosis based on the MTT assay and reduced the Bax/Bcl-2 ratio and the release of cytochrome C based on Western blot analysis. Taken together, our results suggest that Cx43 could mediate resistance against MPP+-induced apoptosis in SH-SY5Y neuroblastoma cells via modulating the mitochondrial apoptosis pathway.

Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

Non-dioxin-like organic toxicant PCB153 modulates sphingolipid metabolism in liver progenitor cells: its role in Cx43-formed gap junction impairment.

The non-dioxin-like environmental toxicant 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), member of a group of persistent organic pollutants wide-spread throughout the environment, reduces gap junction intercellular communication (GJIC), an event possibly associated with tumor promotion. Since very few studies have investigated the signaling effectors and mode(s) of action of PCB153, and it is known that the gap junction (GJ) protein Cx43 can be regulated by the bioactive sphingolipid (SL) sphingosine 1-phosphate (S1P), this in vitro study mainly addresses whether SL metabolism is affected by PCB153 in rat liver epithelial WB-F344 cells. PCB153 treatment obtained significant changes in the S1P/ceramide (Cer) ratio, known to be crucial in determining cell fate. In particular, an increase in S1P at 30min and a decrease of the bioactive lipid at 3h were observed, whereas Cer level increased at 1h and 24h. Notably, a time-dependent modulation of sphingosine kinase (SphK), the enzyme responsible for S1P synthesis, and of its regulators, ERK1/2 and protein phosphatase PP2A, supports the involvement of these signaling effectors in PCB153 toxicity. Electrophysiological analyses, furthermore, indicated that the lipophilic environmental toxicant significantly reduced GJ biophysical properties, affecting both voltage-dependent (such as those formed by Cx43 and/or Cx32) and voltage-independent channels, thereby demonstrating that PCB153 may act differently on GJs formed by distinct Cx isoforms. SphK down-regulation alone induced GJIC impairment, and, when combined with PCB153, the acute effect on GJ suppression was additive. Moreover, after enzyme-specific gene silencing, the SphK1 isoform appears to be responsible for down-regulating Cx43 expression, while being the target of PCB153 at short-term exposure. In conclusion, we provide the first evidence of novel effectors in PCB153 toxic action in rat liver stem-like cells, leading us to consider SLs as potential markers for preventing GJIC deregulation and, thus, the tumorigenic action elicited by this environmental toxicant.

Effect of evodiamine on the gap junction intercellular communication and synthesis of collagen in keloid

Objective To investigate the effect of evodiamine on the proliferation of fibroblasts, synthesis of collagen, and gap junction protein connexin 43 (Cx43) in keloid. Methods The cultured in vitro fibroblasts from patients with keloid were harvested and divided into a control group and evodiamine-treated (0.5, 1.0, 2.0, and 4.0 μmol/L) groups. After treatment for 24, and 48 h, cell activity was determined by methyl thiazol tetrazolium (MTT). The expression of gap junction protein Cx43, and systhesis of collagen type Ⅰ and Ⅲ in vitro after treatment were measured by enzyme linked immunosorbent assay (ELISA). Results At 24 h cell inhibition rate was 6.2%, 9.1%, 12.5%, and 17.6% in 0.5, 1.0, 2.0, 4.0 μmol/L evodiamine-treated groups, respectively. Evodiamine could significantly inhibit the growth of fibroblasts in vitro in a concentration- and time-dependent manner. ELISA results revealed that Cx43 protein absorbance (A) values were 0.252, 0.315, 0.383 and 0.547, those of collagen type Ⅰ were 0.507, 0.412, 0.336 and 0.226, and those of Collagen type Ⅲ were 0.246, 0.228, 0.179 and 0.115 in the 0.5, 1.0, 2.0, 4.0 μmol/L evodiamine-treated experimental groups, respectively. The expression of gap junction protein Cx43 was up-regulated, and the systhesis of collagen type Ⅰ and Ⅲ in vitro was decreased after evodiamine treatment. Conclusion Evodiamine can effectively promote the production of gap junction protein Cx43, increase the level of gap junction intercellular communication (GJIC), inhibit the growth of fibroblasts and the production of collagen type Ⅰ and Ⅲ of keloid in vitro. Key words: Evodiamine; Keloid; Fibroblast; Gap junction intercellular communication; Gap junction protein; Collagen type Ⅰ; Collagen type Ⅲ

Sustained Release of Cx43 Antisense Oligodeoxynucleotides from Coated Collagen Scaffolds Promotes Wound Healing.

Antisense oligodeoxynucleotides targeting the mRNA of the gap junction protein Cx43 promote tissue repair in a variety of different wounds. Delivery of the antisense drug has most often been achieved by a thermoreversible hydrogel, Pluronic F-127, which is very effective in the short term but does not allow for sustained delivery over several days. For chronic wounds that take a long time to heal, repeated dosing with the drug may be desirable but is not always compatible with conventional treatments such as the weekly changing of compression bandages on venous leg ulcers. Here the coating of collagen scaffolds with antisense oligonucleotides is investigated and a way to provide protection of the oligodeoxynucleotide drug is found in conjunction with sustained release over a 7 d period. This approach significantly reduces the normal foreign body reaction to the scaffold, which induces an increase of Cx43 protein and an inhibition of healing. As a result of the antisense integration into the scaffold, inflammation is reduced with the rate of wound healing and contracture is significantly improved. This coated scaffold approach may be very useful for treating venous leg ulcers and also for providing a sustained release of any other types of oligonucleotide drugs that are being developed.

Polymorphism in the C-terminal Cytoplasmic Site, Arginine Phosphorylation Site and 3′UTR of Connexin 43 and its Association with the Progression of Disc Herniation

Introduction Intervertebral disc being an avascular tissue, the gap junction protein connexin play an important role in nutrient and extracellular matrix protein play a major role in its structural stability. The pathophysiology underlying the process of Disc Herniation is still not clear but genetic factors play a vital role in the early occurrence of the disease. The study was initiated to determine the association of Connexin 43 gene polymorphisms and protein levels in Disc herniated patients in comparison to healthy individuals in Indian population. Methods Pre Operative blood samples were collected from 20 patients undergoing surgery for disc herniation between the age group of 20–60years and 10 samples from healthy individuals with no history of Intervertebral disc disease. DNA was isolated, primers designed for exonic and UTR regions of Connexin 43 performed PCR and samples sequenced for analysis of polymorphic variants. Genotypic and phenotypic distributions were compared with Healthy controls. Hardy Weinburg calculations were done to correlate the allelic/genotypic frequencies in the both study groups. Levels of Connexin 43 were assayed by sandwich ELISA by utilizing a standard Streptavidin-HRP format. Results The sequencing of amplified PCR product of connexin 43 resulted in identification of 14 variants in the cytoplasmic region and 6 variants in the 3′UTR. The variant thr326pro, arg362 gln, arg366lys and arg376 gln were statistically significant. In the 3′ UTR there were 6 variants and almost all the herniated samples had 1 or 2 variants with C12508T statistically significant. There were multiple variants in individuals below the age of 40 years. Genetic variants both homozygous as well as heterozygous were more common in lower age groups of 20–40. The levels of connexin 43 were very high in herniated samples as compared with control ( p < 0.0453). Conclusion Connexin43 polymorphisms at amino acid position 362 CGA-CAA (arginine to glutamine) and 376 CGG-CAG (arginine to gluramine) in herniated samples could hinder the hexamerisation of Connexon gap junction channel. The amino acid 362 and 376 are the binding sites for connexon hexamers after phosphorylation at the C- terminal region of the transmembrane protein. The functional connexin 43 is a transmembrane form. This shows that the 3′UTR polymorphism increases their expression but the protein does not get incorporated into the membrane due to polymorphic variants at the site of Arginine phosphorylation (position 362 and 376) and therefore is not physiologically functional. This profound reduction in gap junction channels could cause both nutrient and oxidative stress as the disc tissue is an avascular tissue the mode of channelizing between tissues is via the connexin gap junctions. The 3′UTR variants of connexin 43 can induce an increased expression of the mRNA. This study was further extended to check the levels of the protein in the disc lysates of herniated and control samples by ELISA. The Connexin 43 levels were very high in diseased samples ( p < 0.0453) which can be hypothesized as biophysically nonfunctional gap junctional protein localized in the cytoplasma due to polymorphic variants at the site of Arg phosphorylation site which is one of the post translational requirements for hexamerisation of the connexin 43 and insertion into the membrane as channels. This is the first study to report a SNP associated mutation in gap junction protein Cx43.

Characterizing the Molecular Pathology of Arrhythmogenic Cardiomyopathy in Patient Buccal Mucosa Cells.

Analysis of myocardium has revealed mechanistic insights into arrhythmogenic cardiomyopathy but cardiac samples are difficult to obtain from probands and especially from family members. To identify a potential surrogate tissue, we characterized buccal mucosa cells. Buccal cells from patients, mutation carriers, and controls were immunostained and analyzed in a blinded fashion. In additional studies, buccal cells were grown in vitro and incubated with SB216763. Immunoreactive signals for the desmosomal protein plakoglobin and the major cardiac gap junction protein Cx43 were markedly diminished in buccal mucosa cells from arrhythmogenic cardiomyopathy patients with known desmosomal mutations when compared with controls. Plakoglobin and Cx43 signals were also reduced in most family members who carried disease alleles but showed no evidence of heart disease. Signal for the desmosomal protein plakophilin-1 was reduced in buccal mucosa cells in patients with PKP2 mutations but not in those with mutations in other desmosomal genes. Signal for the desmosomal protein desmoplakin was reduced in buccal mucosa cells from patients with mutations in DSP, DSG2, or DSC2 but not in PKP2 or JUP. Abnormal protein distributions were reversed in cultured cells incubated with SB216763, a small molecule that rescues the disease phenotype in cardiac myocytes. Buccal mucosa cells from arrhythmogenic cardiomyopathy patients exhibit changes in the distribution of cell junction proteins similar to those seen in the heart. These cells may prove useful in future studies of disease mechanisms and drug screens for effective therapies in arrhythmogenic cardiomyopathy.

Irx3 is required for postnatal maturation of the mouse ventricular conduction system.

The ventricular conduction system (VCS) orchestrates the harmonious contraction in every heartbeat. Defects in the VCS are often associated with life-threatening arrhythmias and also promote adverse remodeling in heart disease. We have previously established that the Irx3 homeobox gene regulates rapid electrical propagation in the VCS by modulating the transcription of gap junction proteins Cx40 and Cx43. However, it is unknown whether other factors contribute to the conduction defects observed in Irx3 knockout (Irx3−/−) mice. In this study, we show that during the early postnatal period, Irx3−/− mice develop morphological defects in the VCS which are temporally dissociated from changes in gap junction expression. These morphological defects were accompanied with progressive changes in the cardiac electrocardiogram including right bundle branch block. Hypoplastic VCS was not associated with increased apoptosis of VCS cardiomyocytes but with a lack of recruitment and maturation of ventricular cardiomyocytes into the VCS. Computational analysis followed by functional verification revealed that Irx3 promotes VCS-enriched transcripts targeted by Nkx2.5 and/or Tbx5. Altogether, these results indicate that, in addition to ensuring the appropriate expression of gap junctional channels in the VCS, Irx3 is necessary for the postnatal maturation of the VCS, possibly via its interactions with Tbx5 and Nkx2.5.

HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells.

Human papillomavirus type 16 (HPV16) causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large). Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43), a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G) Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.

The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system.

Cardiomyocytes are electrically coupled by gap junctions, defined as clusters of low-resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx40, Cx43 and Cx45, which are present in cardiomyocytes, is known to be developmentally regulated. This study investigates the premise that alterations in gap junction proteins are one of the mechanisms by which teratogens may act. Specifically, those molecules known to be teratogenic in humans could cause their effects via disruption of cell-to-cell communication pathways, resulting in an inability to co-ordinate tissue development. Caffeine significantly inhibited contractile activity at concentrations above and including 1500 μm (P < 0.05), while not affecting cell viability and total protein, in the embryonic chick cardiomyocyte micromass culture system. The effects of caffeine on key cardiac gap junction protein (Cx40, Cx43 and Cx45) expression were analysed using immunocytochemistry and in-cell Western blotting. The results indicated that caffeine altered the expression pattern of Cx40, Cx43 and Cx45 at non-cytotoxic concentrations (≥2000 μm), i.e., at concentrations that did not affect total cell protein and cell viability. In addition the effects of caffeine on cardiomyocyte formation and function (contractile activity score) were correlated with modulation of Cxs (Cx40, Cx43 and Cx45) expression, at above and including 2000 μm caffeine concentrations (P < 0.05). These experiments provide evidence that embryonic chick cardiomyocyte micromass culture may be a useful in vitro method for mechanistic studies of perturbation of embryonic heart development. Copyright © 2015 John Wiley & Sons, Ltd.

Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells.

Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, mediate the transfer of biological information between donor and acceptor cells. Although the biological effects of exosomes in target cells have been intensively studied, the mechanisms that govern exosomal uptake are not fully understood. Here, we show that Connexin 43 (Cx43), the most widely expressed GJ protein, is present in exosomes in the form of hexameric channels and, more importantly, that exosomal Cx43 is able to modulate the interaction and transfer of information between exosomes and acceptor cells. This study envisions a new paradigm where Cx43-containing channels mediate the release of exosomal content into cells, which constitutes a novel and unanticipated mechanism to modulate intercellular communication.

Abstract 164: Production of De Novo Cardiomyocytes in Adult Asxl2-/- Hearts

The chromatin-associated protein ASXL2 is highly expressed in the heart, where it critically regulates the levels of two repressive histone marks, trimethylated H3 lysine 27 and ubiquitinated H2A. We have previously observed that Asxl2-/- hearts displayed abnormal growth during adulthood, resulting in disproportionally larger heart from 4 months on. This abnormal growth is not due to cardiomyocyte hypertrophy, as demonstrated by measurement of comprehensive dimensions of isolated cardiomyocytes. BrdU labeling showed an elevation in proliferation index in Asxl2-/- hearts at 3 months. At the time of labeling, with rare exceptions, labeled cells are interstitial non-cardiomyocytes in both wildtype control and Asxl2-/- hearts. The vast majority of labeled cells stained positive for vimentin, a mesenchymal marker, and negative for Nkx2.5, Gata4 or c-kit. EdU pulse-chase experiments were performed to determine the fate of labeled cells. After a 4-week chase, a significant number of EdU+/cTnT+ cardiomyocytes were present in Asxl2-/- hearts but not in wildtype control hearts. Expression of the gap junction protein Cx43 was detected between EdU+ cardiomyocytes and neighboring EdU- cardiomyocytes, suggesting electrical coupling. Cardiomyocytes were isolated from pulsed-chased Asxl2-/- hearts for additional morphological analysis. Isolated EdU+/cTnT+ cardiomyocytes vary greatly in cell size, shape and nucleation status, ranging from small, spindle-shaped and mononuclear to large, rod-shaped and binuclear. Taken together, our data suggest that new cardiomyocytes are produced in adult Asxl2-/- hearts from a population of proliferative interstitial cells that are distinct from c-kit+ cardiac stem cells. This raises intriguing possibilities for cardiac repair and regeneration.

Hsp47 mediates Cx43-dependent skeletal growth and patterning in the regenerating fin

Skeletal morphogenesis describes how bones achieve their correct shape and size and appropriately position joints. We use the regenerating caudal fin of zebrafish to study this process. Our examination of the fin length mutant short fin (sof b123) has revealed that the gap junction protein Cx43 is involved in skeletal morphogenesis by promoting cell proliferation and inhibiting joint formation, thereby coordinating skeletal growth and patterning. Here we demonstrate that serpinh1b is molecularly and functionally downstream of cx43. The gene serpinh1b codes for a protein called Hsp47, a molecular chaperone responsible for proper folding of procollagen molecules. Knockdown of Hsp47 in regenerating fins recapitulates the sof b123 phenotypes of reduced fin length, reduced segment length and reduced level of cell proliferation. Furthermore, Hsp47 knockdown affects the organization and localization of the collagen-based actinotrichia. Together, our findings reveal that serpinh1b acts in a cx43 dependent manner to regulate cell proliferation and joint formation. We conclude that disruption of the collagen-based extracellular matrix influences signaling events required for cell proliferation, as well as the patterning of skeletal precursor cells that influences segment length. Therefore, we suggest that Hsp47 function is necessary for skeletal growth and patterning during fin regeneration.

The role of connexin 43 in mediating odor response

Connexin proteins are the hemichannels that form gap junctions to regulate the intercellular communication. Connexin 43 (Cx43) is the most common gap junction protein that expresses in many cell types, including the olfactory sensory neurons. Phosphorylation is a crucial step to regulate the function of Cx43. Gap junction was found to modulate the odor response, but the specific role is still elusive. Here, we report that gap junctions play a role in odor-evoked calcium response in both heterologous cell system and primary olfactory sensory neurons. This regulation is mediated through gap junction protein Cx43. Overexpression of full length Cx43 can counteract the inhibitory effect of gap junction or connexin blockers on odor-evoked [Ca2+]i increase in hana3A cells. Carboxy-terminal of Cx43 (Cx43CT) has the similar function as the full length of Cx43. Furthermore, we found that expression level of phosphorylation of Cx43 at S368 is dynamic with the stimulation of odor in hana3A cells. Expression level of phosphorylated Cx43 at S368 was decreased when gap junction or connexin inhibitors were applied. Phosphorylation of Cx43 during odor or inhibitor stimulation may be mediated by ERK and JNK signaling pathway. Altogether our data suggest that expression of Cx43 can regulate the odor response. This study provides a clue to indicate the possible protective mechanism of gap junction in odor response.

Postnatal Development and Maturation of the Vestibular Organ in Dominant-Negative Connexin26 Transgenic Mouse

Backgrounds: Immunohistochemical studies have revealed that connexin 26 (Cx26) exists not only in the cochlea but also in the vestibular organs. K+ cycling involving gap junction protein Cx26 in the vestibular labyrinth, which is similar to that in the cochlea, is thought to play a fundamental role in endolymph homeostasis and sensory transduction. Methods: We analyzed the morphological and functional development of the vestibular organ in Cx26 transgenic mice between 0 days after birth (P0) and P140, which was compared with that of littermate control mice (non-Tg). Results: The gross structure of the inner ear in non-transgenic and transgenic mice revealed no hydrops, no defects, no degeneration in either the cochlea or the vestibule throughout the postnatal period. Light microscopic observations in the sensory epithelium revealed normally developed and matured utricula macula, saccular macula, and ampulla in the transgenic mice, which were very similar to those of the nontransgenic mouse. Conclusions: The present study clearly demonstrated that postnatal development and maturation in the vestibular organ were morphologically and functionally completed in Cx26 transgenic mice.

The central role of aquaporins in the pathophysiology of ischemic stroke

Stroke is a complex and devastating neurological condition with limited treatment options. Brain edema is a serious complication of stroke. Early edema formation can significantly contribute to infarct formation and thus represents a promising target. Aquaporin (AQP) water channels contribute to water homeostasis by regulating water transport and are implicated in several disease pathways. At least 7 AQP subtypes have been identified in the rodent brain and the use of transgenic mice has greatly aided our understanding of their functions. AQP4, the most abundant channel in the brain, is up-regulated around the peri-infarct border in transient cerebral ischemia and AQP4 knockout mice demonstrate significantly reduced cerebral edema and improved neurological outcome. In models of vasogenic edema, brain swelling is more pronounced in AQP4-null mice than wild-type providing strong evidence of the dual role of AQP4 in the formation and resolution of both vasogenic and cytotoxic edema. AQP4 is co-localized with inwardly rectifying K+-channels (Kir4.1) and glial K+ uptake is attenuated in AQP4 knockout mice compared to wild-type, indicating some form of functional interaction. AQP4-null mice also exhibit a reduction in calcium signaling, suggesting that this channel may also be involved in triggering pathological downstream signaling events. Associations with the gap junction protein Cx43 possibly recapitulate its role in edema dissipation within the astroglial syncytium. Other roles ascribed to AQP4 include facilitation of astrocyte migration, glial scar formation, modulation of inflammation and signaling functions. Treatment of ischemic cerebral edema is based on the various mechanisms in which fluid content in different brain compartments can be modified. The identification of modulators and inhibitors of AQP4 offer new therapeutic avenues in the hope of reducing the extent of morbidity and mortality in stroke.